Project Summary/Abstract Vasopressin (VP) is a key hormone that regulates water balance by stimulating water reabsorption in the kidney. Neuroendocrine VP neurons in the paraventricular (PVH) and supraoptic (SON) hypothalamus are the sole source of circulating VP. Therefore, activity of these neurons must be tightly controlled to achieve water balance. Two temporally distinct signals are known to affect VP neuron activity: 1) systemic signals that convey information about systemic osmolality and intravascular volume, and 2) rapid ?presystemic? signals that inform VP neurons about future anticipated changes in systemic osmolality. Presystemic signals include the detection of cues in the environment that anticipate future water ingestion or the actual ingestion of water or food itself (note that eating leads to an increase in blood osmolality). We have established that presystemic signals related to water/food act in seconds to decrease/increase VP neuron activity, respectively. Systemic regulation of VP release is known to be mediated by three structures in the lamina terminalis (LT), namely, the SFO, MnPO, and OVLT. SFO and OVLT are outside the blood-brain barrier and contain osmosensitive and angiotensin II-responsive neurons that send systemic osmolality information to VP neurons directly or indirectly through MnPO. In contrast, the neural mechanism for presystemic regulation of VP release remains largely unknown. To find the source of presystemic regulation, we have performed a series of monosynaptic rabies mapping and ChR2-assisted circuit mapping studies to identify the afferents of neuroendocrine VP neurons. The major input to VP neurons comes from the three sites in the LT, and these sites provide mainly excitatory inputs to VP neurons. Importantly, our preliminary studies have revealed that SFOVglut2 and MnPO/OVLTVglut2neurons, like VP neurons, also undergo rapid presystemic regulation. Thus, presystemic regulation of VP neurons is likely mediated by the LT. These results lead us to propose the following hypotheses: 1) All three LT structures, SFO, MnPO, and OVLT, contribute to presystemic regulation of VP neurons. 2) The MnPO is the primary site where neural presystemic signals enter the LT. Presystemic signals will then be distributed to the SFO and OVLT. 3) Presystemic regulation of VP release, drinking, and feeding is mediated by the same structure, the LT. The project described here will use combinations of cutting-edge neuroscience techniques, such as rabies mapping, optetrode recordings, in vivo GCaMP imaging, ChR2- assisted circuit mapping, and chemogenetics, to test above hypotheses. We aim to build a simple mechanistic model for systemic and presystemic regulation of water balance, which will provide a comprehensive description of neural mechanisms underlying diverse physiological and behavioral processes affected by water balance, such as VP release, drinking, and feeding.